The American Diabetes Association estimates that there are currently 5 million people in the United States with confirmed diabetes, and over 10 million at risk.
The cost of this disease and its sequelae to the American economy is staggering. Care of diabetics consumes a total of $98 billion per year, accounting for one of every seven healthcare dollars spent in the U.S. There are 24,000 new cases of diabetes-caused blindness caused by diabetes each year. Diabetes is the leading cause of kidney failure, contributing about 40% of new dialysis patients. Diabetes is also the most frequent cause of lower limb amputation, with 56,000 limbs lost to diabetes each year. The per capita health care costs incurred per diabetic person is $10,071 annually, compared with $2,669 for non-diabetics.
Type I diabetes mellitus (also known as insulin-dependent diabetes) is a severe condition accounting for 5-10% all diabetics. The pathology arises because the patient's insulin-secreting beta cells in the pancreas have been eliminated by an autoimmune reaction. Under current practice, the condition is managed by regular injection of insulin, constant attention to diet, and continuous monitoring of blood glucose levels to adjust the insulin dosing. It is estimated that the market for recombinant insulin will reach $4 billion by 2005. Of course, the availability of insulin is life-saving for Type I diabetics. But there is no question that the daily regimen of administration and monitoring that diabetics must adhere to is toublesome to the end user, and not universally effective.
For this reason, there are several clinical tests underway to transplant diabetics with islet cells isolated from donor pancreas. This has been made possible by recent advances in the isolation and culture of islet cells. U.S. Pat. No. 4,797,213 described separation of islets of Langerhans. U.S. Pat. No. 4,439,521 reports a method for producing self-reproducing pancreatic islet-like structures. U.S. Pat. No. 5,919,703 reports preparation and storage of pancreatic islets. U.S. Pat. No. 5,888,816 reports cell culture techniques for pancreatic cells using hypothalamus and pituitary extracts. WO 00/72885 reports methods of inducing regulated pancreatic hormone production in non-pancreatic islet tissues. WO 00/78929 reports methods of making pancreatic islet cells. Kim et al. (Genes Dev. 15:111, 2001) review the intercellular signals regulating pancreas development and function. Yamaoka et al. (Int. J. Mol. Med. 3:247, 1999) review the development of pancreatic islets, and the putative role of factors such as Sonic hedgehog and activin, transcriptional factors like PDX1 and Isl1, growth factors like EGF and HGF, hormones like insulin and growth hormone, and cell adhesion molecules such as N-CAM and cadherins.
Peck et al. (Ann. Med. 33:186, 2001) propose that pancreatic stem cells be used as building blocks for better surrogate islets for treating Type I diabetes. WO 00/47721 reports methods of inducing insulin positive progenitor cells. WO 01/39784 reports pancreatic stem cells isolated from islet cells that are nestin-positive. WO 01/77300 reports human pancreatic epithelial progenitors that are proposed to have the capacity to differentiate into acinar, ductal, and islet cells. Deutsch et al. (Development 128:871, 2001) describe a bipotential precursor population for pancreas and liver within the embryonic endoderm. Zulewski et al. (Diabetes 50:521, 2001) describe multipotential nestin-positive stem cells isolated from adult pancreatic islets that differentiate into endocrine, exocrine, and hepatic phenotypes. U.S. Pat. No. 6,326,201 (Curis Inc.) reports pancreatic progenitor cells made by dissociating and culturing cells from pancreatic duct. The present clinical experience of islet cell transplantation is reviewed by Bretzel et al. (Exp. Clin. Endocrinol. Diabetes 190 (Suppl. 2):S384, 2001) and Oberholzer et al. (Ann. N.Y. Acad. Sci. 875:189, 1999). The current clinical trials typically involve infusing cells from at least two pancreas donors. Even if this treatment proves to be successful, there will be insufficient material available from current sources to treat all the eligible Type I diabetic patients.
Developmental work has been done in several institutions to capitalize on the promise of pluripotent stem cells from the embryo to differentiate into other cell types. Cells bearing features of the islet cell lineage have reportedly been derived from embryonic cells of the mouse. For example, Lumelsky et al. (Science 292:1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Soria et al. (Diabetes 49:157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.
Regrettably, the mouse model of embryonic stem cell development is its own peculiar case, and does not yield strategies for differentiation that are applicable to other species. In fact, pluripotent stem cells have been reproducibly isolated from very few other mammalian species. Only recently did Thomson et al. isolate embryonic stem cells from human blastocysts (Science 282:114, 1998). Concurrently, Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlike mouse embryonic stem cells, which can be kept from differentiation simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very special conditions (U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616). Accordingly, it is necessary to develop completely new paradigms to differentiate human pluripotent cells into fully functional differentiated cell types.
Jacobson et al. (Transplant. Proc. 33:674, 2001) reported differentiation of intestinal and pancreatic endoderm from rhesus embryonic stem cells. Assady et al. (Diabetes 50:1691, 2001) identified insulin production by human embryonic stem cells differentiated to embryoid bodies, by immunohistochemistry and enzyme-linked immunoassay of the culture medium. Of course, embryoid bodies contain an enormous variety of different cell types (WO 01/51616), and Assady made no attempt to isolate the insulin-secreting cells or determine differentiation conditions that would produce enriched populations.
For embryonic stem cell derived islet cells to become a commercially viable proposition, there is a need to develop new procedures that provide for populations of islet cells of high purity.